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Endocannabinoidome Advanced article

Iannotti Fabio Arturo, Istituto di Chimica Biomolecolare Consiglio Nazionale Article Contents
• Introduction
delle Ricerche, Pozzuoli, Italy
• Redundancy and Promiscuity of eCB Metabolic
Piscitelli Fabiana, Istituto di Chimica Biomolecolare Consiglio Nazionale delle Pathways and Enzymes

Ricerche, Pozzuoli, Italy • N-acyl Amino Acids

• N-acyl-dopamines and N-acyl-serotonins
• Products of the Oxidative Metabolism of eCBs
and Related Lipids
• Fatty Acid Esters of Hydroxyl Fatty Acids
• Conclusions: How to Investigate and Manage
the eCBome

Online posting date: 16th November 2018

The classical definition of the endocannabinoid sys- endocannabinoid system (ECS) in numerous diseases and
tem (ECS), at the turn of the century, was that of disorders from this research. Initially, the ECS was defined as
a complex pleiotropic system composed by: (1) the the endogenous lipid signalling system comprised of (1) the
two cannabinoid receptors (CB1 and CB2 ); (2) their two most potent endogenous agonists of cannabinoid recep-
endogenous ligands, the ‘endocannabinoids’ (EC) tors, anandamide (N-arachidonoylethanolamine, AEA) and
and (3) the five enzymes believed at that time to 2-arachidonoyl-glycerol (2-AG), also named endocannabi-
noids; (2) several endocannabinoid-related molecules, of which
be uniquely responsible for EC biosynthesis and
N-oleoylethanolamine (OEA) and N-palmitoylethanolamine
degradation. However, studies carried out during
(PEA) are the most studied; (3) the enzymes regulating the
the last 10 years have revealed the potential exis-
endocannabinoid biosynthesis (NAPE-PLD, ABDH4, GDE1,
tence of a high degree of redundancy for both the
PTPN22 for AEA, and DAGLα and DAGLβ for 2-AG) and
molecular targets and metabolic routes, and cor-
k degradation (FAAH for AEA, and MAGL, ABDH6, ABDH12 k
responding enzymes, of the ECs. Therefore, these and FAAH for 2-AG); and (4) the two endocannabinoids
new discoveries suggested that the time had come responsive GPCRs known as cannabinoid receptor of type
to expand our view of the ECS. In fact, other bioac- 1 (CB1) and cannabinoid receptor of type 2 (CB2) and the
tive long chain fatty acid amides were identified, cation permeant transient receptor potential vanilloid type-1
and their biosynthesis, inactivation and function (TRPV1) (Figure 1a, see also: Endocannabinoid System;
investigated. Since this plethora of novel medi- Endocannabinoid System: An Update; Cannabinoids and
ators has something more than a few chemical Their Receptors). Notably, AEA and 2-AG were also shown to
features in common with ECs, the name of ‘endo- have affinity for noncannabinoid receptors including GABA-A,
cannabinoidome’ (eCBome) was proposed. PPARγ, adenosine A3 and GPR55 (Baggelaar et al., 2018). The
ECS is one of the most pleiotropic signalling systems in verte-
brates by playing a key role in several aspects of the mammalian
physiology and pathology that led researchers to investigate on
Introduction it as it represents a challenging and fascinating target to develop
new therapeutic drugs (see also: Endocannabinoid System;
The discovery of the main psychotropic component of Cannabis Endocannabinoid System: An Update; Cannabinoids and
Sativa, the Δ9 -tetrahydrocannabinol (THC) and the chemical Their Receptors). However, the ECS is more complicated than
synthesis of its analogues provided the foundation for cannabis the one described initially, at the turn of the century. In fact,
research and the following discovery of two G-protein-coupled other endogenous AEA and 2-AG analogues, including other
receptors (GPCRs), named cannabinoid receptors. Therefore, N-acyl-ethanolamines (NAEs), monoacylglycerols, N-acyl amino
growing body of evidence has emerged on the role of the acids, N-acyl-dopamines/taurines/serotonines, were suggested
to be part of this system (Di Marzo and Wang, 2015). Mainly,
because eCBs share biosynthetic and degradative pathways with
eLS subject area: Biochemistry other endogenous signals and are biosynthesised and degraded
through redundant routes (Rahman et al., 2014); moreover, they
How to cite:
Fabio Arturo, Iannotti and Fabiana, Piscitelli (November 2018) may activate other receptors, independently from cannabinoid
Endocannabinoidome. In: eLS. John Wiley & Sons, Ltd: receptors (De Petrocellis and Di Marzo, 2010). These find-
Chichester. ings together with the discovery of new endocannabinoid-like
DOI: 10.1002/9780470015902.a0028301 molecules have led us to expand our view of the eCB system and

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Endocannabinoidome

«Old» view of the endocannabinoid system

sn-1-arachidonate containing
Phosphatidylethanolamine (PE) Phosphatidic acid (PA) Phospholipid
phospolipid

NATs PA phosphohydrolase PLCβ

N-arachidinoyl- sn-1-arachidonate containing

phosphatidylethanolamine (PE) diacylglycerol

ABDH4

PTPN22 NAPE-PLD GDE-1

DAGL α/β

Arachidonate Arachidonate MAGL

O FAAH + + FAAH
Ethanolamide Glycerol OH
OH ABDH6/12 O
N
H OH
O
N-arachidonoylethanolamine (Anandamide, AEA)
2-arachidonoyl-glycerol (2-AG)

CB1 > CB2 TRPV1 CB1 = CB2 TRPV1

(a) Inactivation

The ever expanding «Endocannabinoidome»

Phosphatidylethanolamine (PE) sn-1-arachidonate containing Phosphatidic acid (PA) Phospholipid

phospolipid
k NATs PA phosphohydrolase PLCβ k
sn-1-arachidonate containing
N-arachidinoyl-phosphatidylethanolamine (PE) diacylglycerol
OH
O O
ABDH4 O O P OH
O
O−
N COOH
H
sn-2-arachidonoyl- lysophosphatidic acid (LPA)
PTPN22 NAPE-PLD GDE-1 N-arachidonoyl-glycine

DAGL α/β
OH Kinase
H2N Phosphatase
O
Glycine
MAGL GPR55
O Arachidonic acid Arachidonic acid
FAAH FAAH
OH ABDH6/12 OH
N HO O
H OH
Glycerol
Ethanolamide HO OH O GABAA
Dopamine COX2
N-arachidonoylethanolamine (Anandamide, AEA) COX2 PMs PG-GEs
2-arachidonoyl-glycerol (2-AG)
15-LOX ADENOSINE A3
15 HAEA
OH
O

N OH
GPR55 CB1 > CB2 TRPV1 H PPARγ
CB1 = CB2 TRPV1
N-arachidonoyl-dopamine

EMT EMT
Inactivation
(b) Oxidation

Figure 1 The evolution of the endocannabinoid system from the classical description (a) to the new definition as the ‘endocannabi-
noidome’ (b). ABH4/6/12, αβ-hydrolase 4/6/12; CB1/2, cannabinoid receptor 1/2; COX2, cyclooxygenase 2; DAG, diacylglycerol; EMT, ‘endocannabinoid
membrane transporter’; FAAH, fatty acid amide hydrolase; GDE1, glycerophosphodiester phosphodiesterase 1; GPR55, G-protein-coupled receptor 55;
MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidylethanolamine-selective phosphodiesterase; NATs, N-acyltransferases; PA, phosphatidic acid;
PLCβ, phospholipase Cβ; PLD, phospholipase D; PTPN22, protein tyrosine phosphatase, nonreceptor type 22; TRPV1, transient receptor potential, vanilloid
subtype 1 receptor; GABAA , type-A γ-aminobutyric acid receptors; adenosine A3 , adenosine A3 receptor; PPARγ, peroxisome proliferator-activated receptor
gamma; 15-LOX, lipoxygenase-15; PMs, prostaglandin-ethanolamides/prostamides; PG-GEs, prostaglandin-glyceryl esters.

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Endocannabinoidome

to look at it as a new ‘ome’ in its own right, the ‘endocannabi- 2002), the lipoxygenases-12 and 15 (LOX-12, LOX-15) (Kozak
noidome’ (eCBome, Figure 1b) (Piscitelli et al., 2011; Iannotti and Marnett, 2002) and cytochrome P450 oxygenases (Kozak
et al., 2013; Guida et al., 2018). and Marnett, 2002), known to be involved in eicosanoid pro-
duction from AA (Figure 1b). However, these enzymes can
nevertheless metabolise other members of the two families,
Redundancy and Promiscuity of for example the ω-3 and ω-6 congeners, which are enriched
following certain diets. Moreover, AEA and 2-AG can be pro-
eCB Metabolic Pathways and duced from, and/or catabolised to, other lipid mediators, i.e.
Enzymes sn-2-arachidonoyl-lysophosphatidic acid (LPA) (Fukushima and
Chun, 2001), which acts preferentially on specific GPCRs for
The two first discovered and the most studied eCBs are the LPAs, can act as both biosynthetic precursor and metabolic prod-
AEA or anandamide and the 2-arachidonoylglycerol (2-AG) uct for 2-AG (Nakane et al., 2002) (Figure 1b). These evidences
belonging to the N-acylethanolamine (NAE) and monoacyl- suggested that great care must be taken when manipulating
glycerol (MAGs) families, respectively. AEA and other NAEs pharmacologically the levels of eCB anabolic and catabolic
are biosynthesised via a phospholipid-dependent pathway enzymes. Future studies have to include not only, the observa-
consisting of the enzymatic hydrolysis of the corresponding tion of the consequent phenotypes, but also by the quantitative
N-acyl-phosphatidylethanolamines (NAPEs) (Bisogno et al., analysis of the tissue levels of both the eCBs and their related
2005) (Figure 1). These precursors are generated by transferring mediators.
the acyl group esterified on the sn-1 position of other phos-
pholipids to the nitrogen atom of phosphatidylethanolamine,
via an as-yet unidentified Ca2+ -dependent N-acyl-transferase, N-acyl Amino Acids
although the existence of Ca2+ -independent N-acyl-transferase
suggests the possibility that other enzymes are also capa- N-acyl amino acids encompass a large family of recently
ble of catalysing this reaction. The enzyme catalysing the discovered lipids mediators whose importance has still not
hydrolytic reaction (release of N-acylethanolamine from been fully elucidated. Among them, N-arachidonoyl-l-serine;
N-acyl-phosphatidylethanolamines) was identified as a phos- N-arachidonoyl glycine; N-arachidonoyl taurine, N-oleoyl serine
pholipase D selective for NAPEs (NAPE-PLD), which exhibits and N-oleoyl glycine are the most studied ones.
catalytic properties different from other PLD enzymes. AEA is N-arachidonoyl-l-serine (ARA-S) was isolated for the first time
k one of the least abundant NAEs in comparison to N-stearoyl-, from bovine brain in 2006. In spite of its structural affinity with k
N-palmitoyl- and N-oleoylethanolamine found in tissues and AEA (Figure 2), ARA-S shows very low binding affinity to
cells so far (Iannotti et al., 2016) because arachidonic acid CB1 (<1% of that of AEA) and does not bind CB2 or TRPV1
(AA) is one of the least abundant esterified fatty acids on the receptors (Piscitelli and Bradshaw, 2017). Surprisingly, at least
sn-1 position of phospholipids. Therefore, AEA is biosyn- some of the biological effects exerted by ARA-S resemble those
thesised together with other NAEs, usually more abundant. of AEA and counteracted by cannabinoid receptors antagonists
On the other hand, the major pathway for the biosynthesis of (Kino et al., 2016). To this regard, there are two sequential studies
2-AG comprises the hydrolysis of diacylglycerols containing demonstrating that ARA-S exerts neuroprotective effects follow-
arachidonate moiety in the 2 position (DAGs) catalysed by ing the traumatic brain injury, and this action is dependent on
DAG lipases selective for the sn-1 position (Figure 1). In this TRPV1, but also on CB2 and CB1 receptors and BK K+ chan-
case, the arachidonate moiety is esterified as one of the most nels (Cohen-Yeshurun et al., 2011; Cohen-Yeshurun et al., 2013).
abundant fatty acids. However, 2-AG may be released together In addition, Milman and colleagues demonstrated that ARA-S
with its congener 2-acylglycerols, although these are usually shows similar effects to abnormal cannabidiol (Abn-CBD), a syn-
less abundant. Regarding inactivation pathways, the fatty acid thetic analogous of cannabidiol, which acts as agonist of the third
amide hydrolase (FAAH) recognises as substrates all NAEs and putative cannabinoid receptors (GPR18) producing vasodilator
it also recognises as substrates other long chain fatty acid amides, effects, lowers blood pressure, and induces cell migration, prolif-
including other NAEs, fatty acid primary amides, N-acyl amino eration and mitogen-activated protein kinase (MAPK) activation
acids and N-acyltaurines (Saghatelian et al., 2004; Mulder and in microglia (Piscitelli and Bradshaw, 2017). The biological role
Cravatt, 2006). In some cases, FAAH is also responsible for of N-oleoyl serine (Figure 2) is still mostly unknown except for
2-AG hydrolysis to AA and glycerol (Bisogno et al., 2002), and its role in the bone remodelling by promoting the osteoblast cells
likewise, monoacylglycerol lipase (MAGL) is nonselective for proliferation (Smoum et al., 2010).
2-AG but can hydrolyse also other MAGs. Although, MAGL is N-arachidonoyl glycine (NAGly) is a carboxylic derivative of
the major enzyme responsible for 2-AG degradation in the brain, anandamide (Figure 2). In 2002, (Burstein et al., 2002) demon-
there are other two enzymes, the α/β-hydrolase-6 (ABDH6) and strated that NAGly may serve as an endogenous enhancer of
the α/β-hydrolase-12 (ABDH12), that can inactivate this eCB, tissue anandamide concentrations in vivo. However, this effect
unselectively (Blankman et al., 2007) (Figure 1). The presence was not reproduced in vitro (Piscitelli and Bradshaw, 2017). The
of an arachidonate moiety in both AEA and 2-AG raised the orphan receptors GPR18, GPR72, GPR92 represent the most well
possibility that these molecules might function also as substrate targets of NAGly (Piscitelli and Bradshaw, 2017). Its endoge-
for the same enzymes implicated in AA metabolism. These nous production may occur through two proposed distinct biosyn-
include the cyclooxygenase-2 (COX-2) (Kozak and Marnett, thetic pathways: (1) The first is via an enzymatically regulated

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Endocannabinoidome

O R1
OH
R N R = Alkyl chain
H R1 = Amino acids
O
N-acyl-aminoacids

O COOH O COOH
OH OH
N N
H H

N-arachidonoyl-L-serine (ARA-S) N-oleoyl-serine

O
O OH
OH N
N H
H O
O
N-oleoyl-glycine (OlGly)
N-arachidonoyl-glycine (NAGly)

O
O O O

N S OH N S OH
H H
O O

N-arachidonoyl-taurine N-oleoyl-taurine

Figure 2 Chemical structures of different N-acyl amino acids.

k k
conjugation between the AA and glycine, in a FAAH-dependent N-acyl taurines (Figure 2) were discovered by Saghatelian
reaction. (2) The second one is mediated by an alcohol dehydro- and Cravatt (2005) that identified them in the liver and kid-
genase that produces NAGly as an oxidative metabolite of AEA neys of FAAH(−/−) mice using a large scale mass spectrometry
(Bradshaw et al., 2009). In mammalian, the highest concentra- lipidomics analysis (Saghatelian and Cravatt, 2005). In a subse-
tion of NAGly has been found in the spinal cord and brain. The quent study, the same authors demonstrated that NATs are reg-
biological activity of NAGly is still poorly understood. ulated by FAAH and activate multiple members of the transient
Nevertheless, it has been shown by different research groups receptor potential (TRP) family of calcium channels, including
that NAGly plays an important antinociceptive and antiinflam- TRPV1 and TRPV4 (Saghatelian et al., 2006).
matory role in vivo. However, the exact mechanism of action More recently, N-arachidonoyl taurine and N-oleoyl taurine
through which NAGly exerts these effects remains unknown. The (Figure 2) were demonstrated to possess significant antipro-
affinity for the cannabinoid receptors and TRPV1 is very low liferative effects in human prostate cancer cells (Chatzakos
(Sheskin et al., 1997; Huang et al., 2002; Succar et al., 2007; et al., 2012). Whilst, in pancreatic β-cell lines (HIT-T15) and
Vuong et al., 2008). Another N-acyl glycine that has been most rat islet cell lines (INS-1), N-arachidonoyl-taurines and N-oleoyl
characterised is N-oleoyl glycine (OLGly) (Figure 2). As for taurine resulted in a significant increase in insulin secretion
other N-acyl glycines, OLGLy was detected in partially purified (Waluk et al., 2013), which was then demonstrated by others
rat brain lipid extracts using liquid chromatography combined to imbalance the insulin homeostasis favouring type 2 diabetes
with tandem mass spectrometry (LC/MS/MS) Besides the brain, (Aichler et al., 2017). Finally, Sasso et al. (2016) demonstrated
OLGLy was detected in peripheral organs and tissues including that NATs are novel targets to accelerate the skin repair and
skin, lung, spinal cord, ovaries, kidney, liver and spleen (Piscitelli enhance healing-associated responses in human keratinocytes
and Bradshaw, 2017). OLGly was shown to regulate body tem- and fibroblasts, through a molecular mechanism that involves the
perature and locomotion in rats (Chaturvedi et al., 2006). Most epidermal growth factor receptor (EGFR) and TRPV-1. Finally,
recent evidences demonstrated that OLGLy promote adipogene- in a recent studies, it has been demonstrated that other N-acyl
sis murine 3 T3-L1 cells through the activation of CB1 receptor amino acids including N-docosahexaenoyl-, N-arachidonoyl-
and the enhancement of insulin-mediated Akt signalling path- and N-linoleoyl-GABA, N-docosahexaenoyl-serine,
way (Wang et al., 2015). In addition, OlGly was demonstrated N-docosahexaenoyl-glycine and N-docosahexaenoyl-aspartic
to reduce the nicotine reward and withdraw by directly activat- acid (DAsA), as agonists; and N-docosahexaenoyl proline, bind
ing peroxisome proliferator-activated receptor alpha (PPAR-α) TRPV1 channels as an antagonist (Piscitelli and Bradshaw,
(Donvito et al., 2018). 2017).

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Endocannabinoidome

O OH

R N OH R = Alkyl chain
H
N-acyl-dopamines

OH
OH O
O
N OH
N OH H
H

N-arachidonoyl-dopamine N-oleoyl-dopamine
(NADA) (OLDA)

OH OH
O O

N OH N OH
H H

N-palmitoyl-dopamine N-stearoyl-dopamine
(PALDA) (STEARDA)

Figure 3 Chemical structures of different N-acyldopamines.

N-acyl-dopamines and the bovine brain where it appeared particularly abundant in the
striatum, hippocampus and cerebellum. In contrast, lower levels
k
N-acyl-serotonins of NADA were detected in rat dorsal root ganglia (Huang et al., k
2002). More recent findings showed that NADA is specifically
N-arachidonoyl-dopamine (NADA), expressed in the cell bodies and terminals of dopaminergic
N-Oleoyl dopamine (OLDA), N-palmitoyl neurons. Furthermore, NADA modulates GABAergic neuro-
transmission via a mechanism dependent on both CB1 and
dopamine (PALDA) and stearoyl TRPV1 activation (Piscitelli and Bradshaw, 2017). Interest-
dopamine (STEARDA) ingly, as with the endocannabinoids AEA and 2-AG, NADA is
produced ‘on demand’, responding to specific stimuli.
Among long-chain N-acyl dopamines, the N-arachidonoyl-
In C6 glioma cells, NADA is rapidly taken up by the putative
dopamine (NADA, Figure 3) is the most studied due to of its
endocannabinoid membrane transporter and it is slowly hydrol-
well-characterised activity at TRPV1 and CB1 receptors and its ysed by FAAH to AA and dopamine. However, the affinity of
ability to inhibit T-type Ca2+ channels (Piscitelli and Bradshaw, FAAH for NADA appears much lower than that for AEA, and
2017). The metabolic pathways of NADA as well as of other there is still no evidence of whether the manipulation of FAAH
N-acyl dopamines have not been fully clarified yet. So far, three activity affects NADA levels. On the other hand, NADA is a
alternative biosynthetic pathways have been hypothesised or substrate for O-catecholamine-methyl-transferase (COMT), the
demonstrated. First mechanism consists of the conversion by enzyme involved in catecholamine degradation. COMT converts
aromatic l-amino acid hydroxylase of N-arachidonoyl tyrosine NADA to O-methyl-NADA, that in turn possesses a potency at
(NA-Tyr) to N-arachidonoyl-L-DOPA, which is subsequently least 100 times lower than that of NADA at activating TRPV1
converted to NADA by aromatic l-amino acid decarboxylase, (Huang et al., 2002; Almasi et al., 2008). NADA competes with
although it remains to be established how NA-Tyr is biosyn- [14 C]AEA for the putative endocannabinoid membrane trans-
thesised. The second mechanism, supported by the fact that porter (Ki = 17.3 ± 7.3 μM; compared with Ki = 11.7 ± 1.0 μM
dopamine seems to be necessary to observe the presence of for anandamide inhibition of [14 C]anandamide uptake) (Chu
NADA, consists instead in the direct condensation of AA et al., 2003). This has led to suggest that also NADA can
with dopamine (Hu et al., 2009). Alternatively, an enzyme be taken up by cells through the same mechanisms used for
recently identified in Drosophila melanogaster, arylalkylamine endocannabinoids.
N-acyltransferase-like 2, catalyses the transfer of fatty acid Therefore, due to its peculiarity to activate both CB1 and
chains to the amine group of dopamine (Dempsey et al., 2014). TRPV1, NADA has been proposed to play a role as an endo-
However, no mammalian orthologue of this enzyme has been cannabinoid and as the first endovanilloid with a structure and
identified yet. Also, the mechanisms of NADA catabolism are potency similar to capsaicin. However, the mechanism through
largely unknown. NADA was identified for the first time in which NADA activates CB1 is still not entirely clear. In summary,

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Endocannabinoidome

O NH

R N R = Alkyl chain
H
N-acyl-serotonins OH

O NH
O NH
N
N H
H
OH
N-arachidonoyl-serotonin OH N-oleoyl-serotonin
(AA-5-HT) (OA-5-HT)

O NH O NH

N N
H H

OH OH
N-palmitoyl-serotonin
N-stearoyl-serotonin
(PA-5-HT)

Figure 4 Chemical structures of different N-acylserotonins.

taken together, these studies indicate that NADA behaves as either the treatment of anxiety and stress-related disorders (Micale
a TRPV1 or CB1 agonist depending on its tissue concentration, et al., 2009; Navarria et al., 2014). Only in 2011, however,
and, therefore like AEA, it is believed to be both an endocannabi- Verhoeckx et al. (2011) demonstrated that AA-5H-T and other
k noid and an endovanilloid. N-acyl-serotonins (N-oleoyl-serotonin, N-palmitoyl-serotonin k
N-Oleoyl dopamine (OLDA, Figure 3) is structurally similar to and N-stearoyl-serotonin; Figure 4) are present in the ileum and
NADA, and, although never investigated, its biosynthetic path- jejunum of the gastrointestinal tract of pigs and mice, where they
way is likely similar to that of the latter compound. Detectable act to regulate the glucagon-like peptide-1 (GLP-1) secretion.
levels of this compound, and higher than those of NADA, are Thus, AA-5-HT was the first endogenous antagonist of TRPV1
found in the brain (Huang et al., 2002; Chu et al., 2003). In to be identified, a function only recently extended to oleic acid
a recent study, Zajac and colleagues provided evidence for a at ten-fold higher concentrations (Morales-Lazaro et al., 2016).
dopamine-like catabolic pathway of OLDA, revealing that OLDA Indeed, N-oleoyl-serotonin (OA-5-HT), which is also found in
is methylated by COMT leading to O-methylated products (Zajac the pig and mouse gastrointestinal tract (Verhoeckx et al., 2011),
et al., 2014). OLDA shows affinity for the putative endocannabi- antagonises TRPV1 at micromolar concentrations, similar to
noid membrane transporter in RBL-2H3 cells while being a poor those needed to oleic acid to exert the same effect (Ortar et al.,
substrate for FAAH (Chu et al., 2003). 2007; Morales-Lazaro et al., 2016). Very recently, we showed
Unlike NADA, OLDA, whilst being an agonist at TRPV1, that the levels of AA-5HT and OA-5-HT were significantly
shows very low affinity for CB1. In contrast, two other endoge- reduced in the small intestine in a mouse model of dysbiosis
nous N-acyldopamines, PALDA and STEARDA (Figure 3), were and these alterations might be partly responsible for the effects
found to be inactive per se on TRPV1. However, both these com- of antibiotics and the ensuing dysbiosis on CNS function and
pounds enhanced the effects of NADA on TRPV1 through an subsequent depression-like signs in mice (Guida et al., 2018).
‘entourage’ mechanism comparable to that previously observed Subsequent administration of a probiotic counteracted the
for PEA on AEA and 2-AG (Iannotti et al., 2016). antibiotic-induced behavioural and CNS functional alterations
and concomitantly restored near-physiological levels of intestinal
N-acyl-serotonins N-acyl-serotonins (Guida et al., 2018).

N-arachidonoyl-serotonin (AA-5-HT, Figure 4) was origi-

nally synthetised as a FAAH inhibitor (Bisogno et al., 1998) Products of the Oxidative
and has been shown to increase AEA levels both at periph-
eral and central sites in vivo (Capasso et al., 2005; de Lago
Metabolism of eCBs and Related
et al., 2005). Subsequently, AA-5-HT was found to act as a Lipids
competitive antagonist of TRPV1 receptors, and to be quite
effective, also through this mechanism, in models of both inflam- AA is a substrate of several oxidising agents, as cytochromes
matory and neuropathic pain (Maione et al., 2007) and/or in P-450, lipoxygenases (LOX) and cyclooxygenases (COX)

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R2
O
R = Alkyl chain OH
O O
R2 = -H-CH2-NCH2-OH or -O
OH OH
COX-2 derivatives

O HO
H H OH
N N H
OH OH N
OH
O O
HO O
O HO
OH OH OH
PGE2 ethanolamide (PME2) PGD2 ethanolamide (PMD2) PGF2α ethanolamide (PMF2α)

O HO OH HO OH HO
O O O
OH OH OH
O O O
HO O HO
OH OH OH

k
k

(a) PGE2-glyceryl ester (PGE2-GE) PGD2-glyceryl ester (PGD2-GE) PGF2α-glyceryl ester (PGF2α-GE)

O O
O O O O

OH OH

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Palmitic acid-hydroxy palmitic acid (PAHPA) Palmitic acid-hydroxy stearic acid (PAHSA)

O O
O O O O

OH OH

(b) Oleic acid-hydroxy stearic acid (OAHSA) PalmitOleic acid-hydroxy stearic acid (POHSA)

Figure 5 Chemical structures of different COX-2 derivatives (a) and fatty acid esters of hydroxy fatty acids (b).
Endocannabinoidome

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Endocannabinoidome

that, respectively, introduce one atom, one molecule and two glucose tolerance and reduces adipose inflammation (Syed et al.,
molecules of O2 in the carbon framework. The presence of 2018). Moreover, these effects were GPR40-mediated (Syed
the arachidonoyl group in both 2-AG and AEA makes them et al., 2018).
likely to be oxidised by the same enzymes implicated in the AA
metabolism and producing metabolites analogous to derivatives
produced by AA except for the presence of the glycerol ester
or ethanolamide moiety (Kozak and Marnett, 2002). The most
Conclusions: How to Investigate
interesting oxidative products are those produced by COX-2 for and Manage the eCBome
their biological and pharmacological effects.
COX-2 oxidises 2-AG and AEA, whereas they are oxidised by Given the redundancy and promiscuity of eCB metabolic path-
COX-1 to a much less extent (see also: Cyclooxygenase-2: Biol- ways and enzymes described above, the use of canonical pharma-
ogy of Prostanoid Biosynthesis and Metabolism). They are con- cological tools might not be sufficient to completely distinguish
verted into prostaglandin endoperoxides containing ethanolamide endocannabinoid functions from other endocannabionoid-related
or glycerol functionalities and the most studied are the glyc- molecules. Also, the development of new therapeutic drugs to
erol esters and ethanolamide of PGE2, PGD2, PGF2a and PGI2 treat disorders in which endocannabinoids play a key role might
(Kozak and Marnett, 2002) (Figure 5a). COX-2 is also able be challenging if this complexity is not taken into account. In
to oxidise NAGly, NAla, NAGABA and NADA (Kozak and summary, the eCBome opens new questions:
Marnett, 2002).
It is noteworthy, however, that the biological significance of 1. With so many organ, cellular and molecular targets, how
the aforementioned lipoxydative metabolism is still unclear and do we know if and in what direction the eCB system is
needs to be further investigated. The more simplistic explanation dysregulated during a specific disease, and how can we
would be to classify these pathways just as alternative endo- predict the consequences of such dysregulation?
cannabinoid inactivation modalities. Another, more intriguing 2. With so many biosynthetic enzymes, how do we know which
possibility, would be to consider EC oxidation as an unusual one we need to inhibit in order to reduce eCB tone, and
activation pathway due to the fact that these reactions lead the pro- how do we cope with possible compensatory effects of such
duction of cannabimimetic-derivatives with an enhanced stability redundancy?
compared to their parental endocannabinoids. Moreover, given 3. With so many eCB-like mediators being either produced
that the two main endocannabinoids undergo to different oxida- together, or degraded by the same enzyme as, the eCBs, and
k tive transformations, it might be possible consider that these oxy- so many potential targets being concomitantly dysregulated, k
genated products represent unique signal mediators with potent how do we know that a dysfunctional production or inactiva-
activities distinct from their cannabimimetic precursors or, addi- tion of eCBs is really concurring to a specific disease state,
tionally, that the oxidised endocannabinoids may act as prodrugs and how do we decide that it is really the case to intervene
since these products retain amide or ester functionalities. with?
4. How do we make sure that the intervention is really acting
via the correction of the eCB system?
Fatty Acid Esters of Hydroxyl Fatty
Acids The answer to all these questions is just one: through the tar-
geted profiling of the transcriptional, proteic and metabolic output
The existence of branched fatty acid esters of hydroxy fatty acids of the eCBome in various tissues and organs involved in the
(FAHFAs) was revealed for the first time by Yore et al. (2014) in pathology. This methodology, called ‘endocannabinoidomics’,
serum and many tissues in humans and rodents and was found to and extensively reviewed elsewhere (Piscitelli and Bradshaw;
be associated with type 2 diabetes. In particular, they were able Piscitelli; Bisogno Piscitelli and Di Marzo), could allow us to
to identify 16 FAHFA family members by using an untargeted shed new light into the study of this complex system and could lay
lipidomics approach, including palmitic acid-hydroxy palmitic the foundations for the development of novel cannabinoid-based
acid (PAHPA), oleic acid-hydroxy stearic acid (OAHSA), palmi- drugs.
toleic acid-hydroxy stearic acid (POHSA) (Figure 5b) and oleic
acid-hydroxy palmitic acid (OAHPA). In 2015, an in silico tan- Glossary
dem MS/MS library was developed to allow users to anno-
tate FAHFAs from accurate tandem mass spectra easily and the Endocannabinoid system A complex signalling system
method was validate by identified new FAHFAs species in egg composed by the two cannabinoid receptors (CB1 and CB2),
yolk (Ma et al., 2015). Lee et al. (2016) demonstrated that a their endogenous ligands, named endocannabinoids, and the
specific family of FAHFA, the branched palmitic acid esters of large plethora of enzymes regulating the metabolism of these
hydroxystearic acids (PAHSAs, Figure 5b), has beneficial effects latter.
in murine colitis by exerting antiinflammatory effects partially Endocannabinoidome The plethora of novel identified
regulated by GPR120 (Lee et al., 2016). Very recently, a new molecules that have chemical and functional similarity with
study clearly demonstrated that chronic treatment of chow-fed classical endocannabinoids and that can affect with their
and HFD-mice with PAHSAs enhances insulin sensitivity and activity.

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Endocannabinoidome

N-acylaminoacids Class of endogenous fatty acids injury by reducing apoptosis. Journal of Cerebral Blood Flow &
characterized by a fatty acyl group linked to a primary amine Metabolism 31 (8): 1768–1777.
metabolite by an amide bond. Cohen-Yeshurun A, Willner D, Trembovler V, et al. (2013)
N-acyl-dopamines Class of endogenous fatty acids N-arachidonoyl-L-serine (AraS) possesses proneurogenic proper-
characterized by a fatty acyl group linked to dopamine. ties in vitro and in vivo after traumatic brain injury. Journal of
N-acyl-serotonins Class of endogenous fatty acids Cerebral Blood Flow & Metabolism 33 (8): 1242–1250.
De Petrocellis L and Di Marzo V (2010) Non-CB1, non-CB2
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receptors for endocannabinoids, plant cannabinoids, and synthetic
cannabimimetics: focus on G-protein-coupled receptors and tran-
sient receptor potential channels. Journal of Neuroimmune Phar-
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